**5. Substrates and metabolism**

The metabolism of carbon during fermentation process towards hydrogen is based on transformation of pyruvate in presence of majority of microorganisms active in this reaction. The first step of dark fermentation is based on glycolysis occurying in cytosol of cell, also known as **Embden-Mayerhof-Parnas** (EMP) pathway (Stryer, 1999). This pathway is

There are two main inhibitors of nitrogenase during hydrogen photobiogeneration: molecular oxygen and nitrogen. In the presence of molecular nitrogen occurs competitive nitrogen fixation reaction and this stops almost completely hydrogen evolution. Ammonium ions at concentrations higher than 20 μmol are successful but reversible inhibitors of hydrogen generation (Waligórska, 2009) as well. The nitrogen necessary for the cell

functioning is usually provided by ethanolamine and glutamate.

A. Blue lines - two chains of α tetramer, yellow lines – β subunits, green – P group,

B. P group composed from two subunits of [4Fe-4S]. Colors description the same as in Figure 7. C. Mo-Fe coenzyme. Mo bonded with homocitrate of histidine, Fe bonded with cysteine.

However, glutamate can be the source of nitrogen inhibiting hydrogen evolution similarly as non-ammonium compounds. It can when glutamate becomes the source of carbon after the other sources are exhausted (Koku, 2002). In order to avoid such situation a medium with a relatively high ratio of organic carbon to nitrogen should be applied (e.g. malate to

The metabolism of carbon during fermentation process towards hydrogen is based on transformation of pyruvate in presence of majority of microorganisms active in this reaction. The first step of dark fermentation is based on glycolysis occurying in cytosol of cell, also known as **Embden-Mayerhof-Parnas** (EMP) pathway (Stryer, 1999). This pathway is

red – Fe-Mo coenzyme.

glutamate =15/2 (Eroglu, 1999).

**5. Substrates and metabolism** 

Fig. 8. Dinitrogenase construction (Berg, 2002).

2H+ + 2e + 4ATP → H2 + 4ADP +4Pi (3)

initiated by one molecule of glucose, catalyzed by different enzymes and further transformed into 2 molecules of pyruvate. The energy liberated during oxidation of 3 phosphoglycerol aldehyde is sufficient for phosphorylation of generated acid towards 1,3 bisphosphoglycerol and reduction of NAD+ to NADH. This reaction is catalyzed by 3 phosphoglycerol dehydrogenase. Transformation of glucose to pyruvate is during glycolysis is accompanied by formation two molecules of ATP and two molecules of NADH.

Glucose is not the only substrate in glycolysis. Simple sugars such as fructose or galactose as well as complex sucrose – saccharose, lactose, maltose, cellobiose or cellulose can be used as the initial substrate for glycolysis. However, the incorporation of these complex sugars into glycolysis pathway require initial hydrolysis to the simple carbohydrates.

Glycerol can be considred as a good substarate for glycolysis. A part of glycerol is oxidized into dihydroxyacetone by glycerol dehydrogenase. Next, dihydroxyacetone is phosphorylated into phosphodihydroxyacetone in the presence of dihydroxyacetone kinase. Thanks to triozophosphate isomerase phosphodihydroxoacetone is transformed into 3 phosphoglycerol aldehyde and further participate in EMP pathway.

There are known also other anaerobic pathways transforming glucose into pyruvate as e.g. Entner-Daudoroff or phosphate pentose pathway (Schlegel, 2003, Dabrock, 1992, Vardar-Schara, 2008, Chin, 2003).

**Entner-Doudoroff** pathway goes from glucose to pyruvate and is known also as 2-keto-3-detoxy-6-phosphogluconate. Here, glucose-6-phosphate is transformed with phosphogluconate dehydrogense into 6-phosphogluconate. In the next step, the removal of water from 6-phosphogluconate leads to formation of 2-keto-3-deoxy-6-phosphogluconate. This process is followed by formation of pyruvate and 3-phosphoglycol phosphate. These transformations are analogous to glycolitic pathway already described. One molecule of glucose is transformed into molecules of pyruvate with simultaneous formation of one NADPH (reduced dinucleotide nicotinoamine adenine phosphate and one molecule of ATP (Schlegel, 2003).

**Pentophosphate pathway** is based on initial phosphorylation of glucose to glucose-6 phosphate with help of hexokinase. Further steps are more complicated. The glucose-6 phosphate dehydrogenaze transfer hydrogen to NAD simultaneously forming of gluconolactone. The phosphate gluconolactone dehydrogenase helps to generate 6 phosphogluconate acid. The last phase is based on decarboxylation of the acid into ribuloso-6 phosphate. The transfer of this compound into riboso-5-phosphate and xylulose-5-phosphate starts a non-oxidative phase. At this stage of reaction the reversible reaction between these compounds occurs with formation of sedoheptulose-7-phosphate and 3-phosphoglycerate aldehyde. Subsequent reactions can generate fructose-6-phosphate an erythrose-4-phosphate. In further reactions erythrose-4-phosphate is transformed into 3-phosphoglycerate aldehyde and fructose-6-phosphate. Thus, one cycle of pentophosphate pathway generates 2 molecules of fructose-6-phosphate, one molecule of 3-phosphateglycerol aldehyde, 3 molecules of CO2 and 6 molecules of NADPH. The pentophosphate pathway with glycolysis leads finally to the pyruvate formation (Schlegel, 2003).

In the next steps in anaerobic conditions, the oxidative decarboxylation of pyruvate occurs with acetylo-CoA and CO2 formation. This reaction is catalyzed by pyruvate oxyreductase

Microbiological Methods of Hydrogen Generation 235

or to formate by pyruvate formate lyase (PFL). Hydrogen is produced from formate by the formate hydrogen lyase (FHL) system consisting of hydrogenase 3 (Hyd 3) and formate dehydrogenase-H (FDHH); the FHL is activated by FhlA that is regulated by Fnr and repressed by HycA. Evolved hydrogen is consumed through the hydrogen uptake activity of hydrogenase 1 (Hyd 1) and hydrogenase 2 (Hyd 2). Formate is exported by FocA and/or FocB and is metabolized by formate dehydrogenase-N (FDHN; FdnG), which is linked with nitrate reductase A (NarG) and formate dehydrogenase-O (FDHO; FdoG). HypABCDEF are

Transformation of pyruvate to acetylo-CoA and formic acid occurs in the presence of puruvate-formate liase with relatively anaerobic microorganisms. Formic acid is then transformed into hydrogen and CO2 in the presence of formic-hydrogen lyase. Here, 2 molecules of hydrogen from one molecule of glucose can be generated. Similarly as in the case of completely anaerobic bacteria, pyruvate can form lactic acid (reaction 9), whereas acetylo-CoA into ethanol and acetic acid (reactions 8 and 4). These processes can lower the theoretical amounts of generated hydrogen. Additional negative effect comes from the formation of succinic acid. Namely, formate-hydrogen lyase. become active only at low values of pH what in consequence is caused by formation of acids. Thanks to the decomposition of formic acid further fermentation towards other acids can proceed

The absence of photosystem II in purple non-sulphur bacteria eliminates the problem of oxygen inhibition in hydrogen generation. However, in order to decompose water molecule and generate an electron in the photobiological process, the PNS bacteria need simple organic and inorganic compounds for photosynthesis. Organic compounds are a source of carbon and electrons. The PNS bacteria can use also CO2 as a source of carbon after transformation of metabolism into photoautotrophic one. However, if the light intensity is too low to reduce CO2 then the cell can use H2 and even H2S (at low concentrations) as a source of electrons (Kars, 2010). However, CO2 absorption is the basic metabolic process in the cell developing either in autotrophic or heterotrophic systems. The removal of RuBisCO enzyme *via* genetic modification of PNS bacteria results in the decline of photoheterotrophic development (Akkerman, 2002). Hydrogen generation with PNS bacteria can be realized in the presence of such simple organic molecules as acetate, lactate, malate or glucose. The maximum theoretical yields of conversion of these compounds to photogenerated hydrogen

(acetic acid) C2H4O2 + 2H2O → 2CO2 + 4H2 (10)

(lactic acid ) C3H6O3 + 3H2O → 3CO2 + 6H2 (11)

(malic acid) C4H6O5 + 3H2O → 4CO2 + 6H2 (12)

 (glucose) C6H12O6 + 6H2O → 6CO2 + 12H2 (13) The theoretical amounts are usually much higher than those observed in experiments. The conversion of lactate and malate occurs easily with relatively high yields, but that of acetate and glucose is much more difficult and gives low yields of hydrogen (Kars, 2010). The discrepancies between theoretical values in hydrogen productivity and those obtained in experiments can be explained by different metabolic pathways of carbon in PNS bacteria (Figure 10, Koku, 2002).

maturation proteins for hydrogenases 1, 2, and 3 (Maeda, 2008)

(Dabrock, 1992, Hallenbeck, 2009).

are described by the following equations:

and the reduced form of ferredoxin appears as a step in final oxidation catalyzed by hydrogenase. Here, electrons reduce protons to molecular hydrogen. The reduced ferredoxin is also formed in glycolysis as the result of NADH oxidation to NAD (Dabrock, 1992). Carbon dioxide, acetic acid, lactic acid ethanol, butanol and acetone accompany hydrogen formation:

$$\rm C\_6H\_{12}O\_6 + 2H\_2O \rightarrow 2CH\_3COOH + 2CO\_2 + 4H\_2 \tag{4}$$

$$\rm C\_6H\_{12}O\_6 + 2H\_2O \rightarrow CH\_3COCH\_3 + 3CO\_2 + 4H\_2 \tag{5}$$

$$\text{C}\_{\text{H}}\text{H}\_{12}\text{O}\_{6} \triangleq \text{CH}\_{3}\text{CH}\_{2}\text{CH}\_{2}\text{CH}\_{2}\text{COOH} + 2\text{CO}\_{2} + 2\text{H}\_{2} \tag{6}$$

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 \triangleq \text{CH}\_3\text{CH}\_2\text{CH}\_2\text{CH}\_2\text{OH} + 2\text{CO}\_2 + \text{H}\_2\text{O} \tag{7}$$

$$\rm C\_6H\_{12}O\_6 \to 2CH\_3CH\_2OH + 2CO\_2 \tag{8}$$

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 \xrightarrow{} 2\text{CH}\_3\text{CHOH}\text{COOH} \tag{9}$$

These reactions indicate that theoretical yield of hydrogen should 4 moles of hydrogen per one of glucose when acetone or acetic acid are among the products (Vardar-Schara, 2008).

Fig. 9. Scheme of fermentative hydrogen production in *E. coli* (Maeda, 2008).

Cells metabolize glucose into phosphoenolpyruvate, pyruvate, and formate. Phosphoenolpyruvate is converted to succinate by fumarate reductase (FrdC), and pyruvate is converted to either lactate by lactate dehydrogenase (LdhA), to carbon dioxide (CO2) and acetate by pyruvate oxidase (PoxB), to carbon dioxide by pyruvate dehydrogenase (AceE),

and the reduced form of ferredoxin appears as a step in final oxidation catalyzed by hydrogenase. Here, electrons reduce protons to molecular hydrogen. The reduced ferredoxin is also formed in glycolysis as the result of NADH oxidation to NAD (Dabrock, 1992). Carbon dioxide, acetic acid, lactic acid ethanol, butanol and acetone accompany

C6H12O6 + 2H2O 2CH3COOH + 2CO2 + 4H2 (4)

C6H12O6 + 2H2O CH3COCH3 + 3CO2 + 4H2 (5)

C6H12O6 CH3CH2CH2CH2COOH + 2CO2 + 2H2 (6)

C6H12O6 CH3CH2CH2 CH2OH + 2CO2 + H2O (7)

C6H12O6 2CH3CH2OH + 2CO2 (8)

 C6H12O62CH3CHOHCOOH (9) These reactions indicate that theoretical yield of hydrogen should 4 moles of hydrogen per one of glucose when acetone or acetic acid are among the products (Vardar-Schara, 2008).

Fig. 9. Scheme of fermentative hydrogen production in *E. coli* (Maeda, 2008).

Cells metabolize glucose into phosphoenolpyruvate, pyruvate, and formate. Phosphoenolpyruvate is converted to succinate by fumarate reductase (FrdC), and pyruvate is converted to either lactate by lactate dehydrogenase (LdhA), to carbon dioxide (CO2) and acetate by pyruvate oxidase (PoxB), to carbon dioxide by pyruvate dehydrogenase (AceE),

hydrogen formation:

or to formate by pyruvate formate lyase (PFL). Hydrogen is produced from formate by the formate hydrogen lyase (FHL) system consisting of hydrogenase 3 (Hyd 3) and formate dehydrogenase-H (FDHH); the FHL is activated by FhlA that is regulated by Fnr and repressed by HycA. Evolved hydrogen is consumed through the hydrogen uptake activity of hydrogenase 1 (Hyd 1) and hydrogenase 2 (Hyd 2). Formate is exported by FocA and/or FocB and is metabolized by formate dehydrogenase-N (FDHN; FdnG), which is linked with nitrate reductase A (NarG) and formate dehydrogenase-O (FDHO; FdoG). HypABCDEF are maturation proteins for hydrogenases 1, 2, and 3 (Maeda, 2008)

Transformation of pyruvate to acetylo-CoA and formic acid occurs in the presence of puruvate-formate liase with relatively anaerobic microorganisms. Formic acid is then transformed into hydrogen and CO2 in the presence of formic-hydrogen lyase. Here, 2 molecules of hydrogen from one molecule of glucose can be generated. Similarly as in the case of completely anaerobic bacteria, pyruvate can form lactic acid (reaction 9), whereas acetylo-CoA into ethanol and acetic acid (reactions 8 and 4). These processes can lower the theoretical amounts of generated hydrogen. Additional negative effect comes from the formation of succinic acid. Namely, formate-hydrogen lyase. become active only at low values of pH what in consequence is caused by formation of acids. Thanks to the decomposition of formic acid further fermentation towards other acids can proceed (Dabrock, 1992, Hallenbeck, 2009).

The absence of photosystem II in purple non-sulphur bacteria eliminates the problem of oxygen inhibition in hydrogen generation. However, in order to decompose water molecule and generate an electron in the photobiological process, the PNS bacteria need simple organic and inorganic compounds for photosynthesis. Organic compounds are a source of carbon and electrons. The PNS bacteria can use also CO2 as a source of carbon after transformation of metabolism into photoautotrophic one. However, if the light intensity is too low to reduce CO2 then the cell can use H2 and even H2S (at low concentrations) as a source of electrons (Kars, 2010). However, CO2 absorption is the basic metabolic process in the cell developing either in autotrophic or heterotrophic systems. The removal of RuBisCO enzyme *via* genetic modification of PNS bacteria results in the decline of photoheterotrophic development (Akkerman, 2002). Hydrogen generation with PNS bacteria can be realized in the presence of such simple organic molecules as acetate, lactate, malate or glucose. The maximum theoretical yields of conversion of these compounds to photogenerated hydrogen are described by the following equations:


The theoretical amounts are usually much higher than those observed in experiments. The conversion of lactate and malate occurs easily with relatively high yields, but that of acetate and glucose is much more difficult and gives low yields of hydrogen (Kars, 2010). The discrepancies between theoretical values in hydrogen productivity and those obtained in experiments can be explained by different metabolic pathways of carbon in PNS bacteria (Figure 10, Koku, 2002).

Microbiological Methods of Hydrogen Generation 237

Although the large variety of substrates can be used by photosynthetic bacteria, only a few fulfill the requirements for fast reaction rate and high yield of photogenerated H2. In general, the preferred substrates as anions of organic acids, whereas carbohydrates do not

Biological methods of hydrogen generation from water in presence of microalgae and cyanobacteria are known since seventies of XX century. This process can performed in direct

Cells of certain algae (eg. *Chlamydomonas reinhardtii, Chlorealla fusca*) or cyanobacteria are

This process require absolutely anaerobic conditions. Light energy with wavelength lower than 680 nm is absorbed by photosystem II (PSII) and generate stream of electrons and protons originating from water. Other photosystem (PSI) is induced with light wavelength lower than 700 nm. This allows for transportation of electrons from PSII to PSI *via* chain of reductors called cytochrome *bf*. Electrons from PSI system are transferred *via* ferrodoxine to hydrogenase (algae or cyanobacteria) or nitrogenase (cyanobacteria) and these enzymes reduce protons to molecular hydrogen. In direct biophotolysis neither CO2 nor liqid metabolites are observed. Hydrogenase is very sensitive to oxygen and irreversibly inhibits its activity, therefore constant removal of oxygen is required (Das, 2008). Recent studies concentrate on elimination of sensitivity of algae towards oxygen

This process can be performed with certain cyanobacteria (e.g. *Anabeana variabilis*) in two

12H2O + 6CO2 + light energy → C6H12O6 + 6O2 (15)

 C6H12O6 + 12H2O +light energy → 12H2 + 6CO2 (16) Different periods of oxygen and hydrogen generation allows to eliminate the inhibiting effect of oxygen on enzymes. Similarly, as in direct biophotolysis, photons activate PSI and PSII. In the presence of RuBisCO enzyme the CO2 adsorption occurs, what in consequence of photosynthetic reactions generate glucose and oxygen . In the second step in presence of hydrogense and nitrogenase (Kars, 2009) the decomposition of organic

This type of metabolism in industry is difficult to perform because of periodicity of process. The hydrogen yields generated either by direct or indirect photolysis are unfortunately very

low in comparison with other fermentative methods (Das, 2008).

2H2O + light energy → 2H2 + O2 (14)

capable to split water into molecular hydrogen and oxygen under illumination.

meet the above criteria (Koku, 2002).

and undirect biophotolysis.

**Direct biophotolysis** 

(Benemann, 1997).

compound occurs.

steps:

**Indirect biophotolysis** 

**6. Microbiological methods of hydrogen generation** 

The amount of electrons generated on absorption of organic compounds depends on the source of organic carbon. Even a slight difference in the molecular structure can lead towards completely different metabolic pathway. For example, D- and L-isomers of malate (after conversion into pyruvate) can easily join the TCA cycle. In this way the energy demand for hydrogen generation is met, whereas such a substrate as acetate is used in the other metabolic pathways: e.g. glyoxylate cycle, citramalate cycle, and ethylmalonyl-CoA pathway (Kars, 2010). The excess of electrons generated during assimilation of such substrates as glycerol or butyrate must be accepted during CO2 photoreduction. Therefore, when the only source of carbon is glycerol, it is not assimilated in significant amounts, which is changed after supplementation of glycerol with malate. Initially, malate is assimilated from the medium and evolution of CO2 occurs. In the second step of reaction, the evolved CO2 permits the use of glycerol as a substrate (Pike, 1975).

Fig. 10. Simplified scheme of carbon metabolism in *Rhodobacter sphaeroides* bacteria (Kotay, 2008).

Although the large variety of substrates can be used by photosynthetic bacteria, only a few fulfill the requirements for fast reaction rate and high yield of photogenerated H2. In general, the preferred substrates as anions of organic acids, whereas carbohydrates do not meet the above criteria (Koku, 2002).
